HSV-1/2; obesogen; stool softener; sulfated surfactant

Inactivation of HSV [1]
The in vitro inactivations of HSV by Docusate were compared with another surfactant, SDS. Various concentrations of testing drug were pre-mixed with HSV-1 or HSV-2 and incubated at 37 °C for 1 h. Antiviral effects were then determined by plaque reduction assays (Fig. 1). Inactivation of HSV-1 by Docusate along with SDS is shown in Fig. 1A and that of HSV-2 in Fig. 1B. Results showed that at a concentration of 0.005%, docusate completely inactivated both HSV-1 and HSV-2 after pre-mixing for 1 h (EC90–100=0.005%). In contrast, although this concentration was also effective for SDS, it caused 50% cell death (toxic to cells). At concentration of 0.01%, both docusate and SDS were shown to be toxic to the cells causing 50–100% cell death (data not shown). In these experiments, docusate was slightly more effective against HSV-1 than HSV-2, and slightly more potent and less toxic than SDS at other concentrations (Fig. 1).

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The kinetics of inactivation of HSV by Docusate [1]
To examine the kinetics of inactivation of HSV, docusate at various concentrations was mixed with HSV-2 and incubated at 37 °C in a time-course, with endpoints determined by plaque reduction assay. Results (Fig. 2) showed that docusate had a slight inactivation effect upon HSV-2 at concentrations of 0.001 and 0.0025% after 4 h incubation. However, inactivation reached 50% at time 0, almost 90% (EC90) after 30 min, and 100% after 1 h incubation at a concentration of 0.005%. A concentration of 0.01% of docusate was found to be toxic to cells.

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Short and long incubation cytotoxicities [1]
Microbicidal effectiveness of surface active agents such as Docusate against enveloped viruses suggests a potentially disruptive effect on cellular membranes. Accordingly, experiments were carried out to evaluate relative cytotoxicities of docusate over time. Cytotoxicities of docusate were compared with SDS and measured using Vero cells. Trypan blue exclusion (data not shown) and uptake of neutral red dye were then used to determine the viabilities of cells after incubations with different concentrations of docusate, and SDS. Results were consistent between assays (data not shown). For short term incubation, cells were exposed to docusate for 1 (Fig. 3A) and 6 h (data not shown), whereas for long term incubation, cells were exposed to docusate for 2 (data not shown) and 3 days (Fig. 3B). As can be seen from these figures, after 1 h exposure to docusate and SDS, minimal cytotoxicity of docusate to Vero cells was observed even at concentration of 0.01%. Cytotoxicity was increased after 6 h exposure (data not shown). Concentration at 0.01% was again found to be toxic to Vero cells after 3 days incubation. Thus, the cytotoxicity of docusate is time- and dose-dependent. Furthermore, docusate was slightly less cytotoxic than SDS. CC50 (cytotoxic concentration giving 50% of cell death) of docusate after 2 days incubation was approximately 0.01% and that of SDS was approximately 0.005%.

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Antiviral activity of Docusate determined by pre-treated cells [1]
Some compounds can be internalized into cells or bound to the cellular membranes to exert antiviral effects. This experiment was designed to examine the effect of Docusate on pre-treated cells. Two approaches were employed. First, we pre-incubated Vero cells with docusate and then infected these cells with HSV. In the second, we pre-incubated cells with docusate and then washed it off three times with PBS. The results, together with comparison of SDS, were summarized in Table 1. Cells pre-treated with docusate at a concentration of 0.005%, showed virus infection to be reduced by 45%, and 35% after docusate was removed by three washes. However, this concentration was not able to completely stop virus infection.

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Effect of Docusate on HSV-infected cells [1]
To examine the effect of Docusateon HSV-infected cells, cells were first infected with HSV-2 and then treated with different concentrations of docusate following adsorption by inclusion in the methylcellulose overlay. The results (Table 2) indicated that docusate only had slight inhibitory effect on HSV-infected cells, by approximately 30% plaque reduction at 0.005% of concentration.

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Effect of Docusate on HSV-drug-resistant mutants [1]
To test the antiviral effect of Docusate on the drug-resistant HSV strain, delta 333 virus was pre-mixed with different doses of docusate at 37 °C for 1 h. The antiviral effect was subsequently determined by plaque reduction assay. The results showed that docusate at concentration of 0.005% could completely inactivate delta 333 after incubation at 37 °C for 1 h (Table 3). Although SDS was slightly less efficacious in this assay, it was also capable of inactivating the resistant strain, delta 333. [1]
The effect of Docusate on the polymerase mutant virus was examined by pre-mixing docusate at various doses with 615.8 virus for 1 h at 37 °C. The antiviral activity against polymerase mutant virus was then determined by plaque reduction assay. The results showed that docusate at concentration of 0.005% could inactivate approximately 98% of polymerase mutant 615.8 after incubation at 37 °C for 1 h (Table 4), whereas SDS was less efficacious at this concentration.

Evidence indicates that obesity can be promoted by chemical 'obesogens' that drive adiposity, hunger, inflammation and suppress metabolism. Docusate/Dioctyl sodium sulfosuccinate (DOSS), a lipid emulsifier and candidate obesogen in vitro, is widely used in processed foods, cosmetics and as stool softener medicines commonly used during pregnancy. In vivo testing of Docusate/DOSS was performed in a developmental origins of adult obesity model. Pregnant mice were orally administered vehicle control or Docusate/DOSS at times and doses comparable to stool softener use during human pregnancy. All weaned offspring consumed only standard diet. Adult male but not female offspring of DOSS-treated dams showed significantly increased body mass, overall and visceral fat masses, and decreased bone area. They exhibited significant decreases in plasma adiponectin and increases in leptin, glucose intolerance and hyperinsulinemia. Inflammatory IL-6 was elevated, as was adipose Cox2 and Nox4 gene expressions, which may be associated with promoter DNA methylation changes. Multiple significant phospholipid/sterol lipid increases paralleled profiles from long-term high-fat diet induced obesity in males. Collectively, developmental DOSS exposure leads to increased adult adiposity, inflammation, metabolic disorder and dyslipidemia in offspring fed a standard diet, suggesting that pharmaceutical and other sources of DOSS taken during human pregnancy might contribute to long-term obesity-related health concerns in offspring [2].

Virus plaque reduction assay [1]
Antiviral effects of Docusate were determined by modified plaque reduction assays. Confluent cells were washed with PBS and subsequently infected with HSV for 1 h at 37 °C. After viral inoculum was removed, the infected cells were washed with PBS and overlaid with 0.5% methylcellulose in culture medium. Cells were incubated at 37 °C for 2 days for HSV-2 infection and 3 days for HSV-1. When plaque size was adequate, cells were fixed with 10% formalin and subsequently stained with 0.5% crystal violet. [1]
All data were generated from duplicate or triplicate wells in two or three independent experiments. Mean plaque counts are shown in the tables and figures. Effects of compounds at varying concentrations were expressed as percentage of control (the mean plaque counts in drug treated wells/the mean plaque counts in control wells).

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Effect of Docusate on HSV drug-resistant strains [1]
The antiviral effect of Docusate on HSV-2 thymidine kinase mutant Delta 333 and HSV-1 DNA polymerase mutant 615.8 was performed in a similar way as above (see Section 2.4)

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Inactivation of HSV by Docusate [1]
HSV G and F strains were diluted to 200 pfu/ml with 5% MEM, respectively. Docusate and SDS were diluted to 2× final concentrations (final concentrations: 0, 0.0005, 0.001, 0.0025, 0.005 and 0.01%, respectively) with 5% MEM. Equal volumes of diluted virus and drugs were mixed and incubated at 37 °C water-bath for 1 h. One milliliter of the mixture was then used to infect confluent Vero cells in 6-well plates at 37 °C for 1 h. After infection, viral inoculum was removed and the cells washed with PBS. The cells were subsequently overlaid with 1.5 ml of 0.5% methylcellulose in culture medium for plaque assay. [1]
To examine the kinetics of inactivation of HSV by Docusate, 200 pfu/ml of HSV-2 was pre-mixed with 2× final concentrations of docusate (final concentrations: 0, 0.001, 0.0025, 0.005 and 0.01%, respectively) in 5% MEM and incubated at 37 °C for 0, 15, 30, 60, 120, and 240 min. At each time point, the treated mixture was used to infect confluent Vero cells. After viral inoculum was removed, the cells were covered with methylcellulose for plaque assay.

Docusate Stock solutions (1%, w/v) was prepared by dissolving them in sterile deionized distilled water and stored at room temperature.

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Cytotoxicity of Docusate [1]
The cytotoxicity of Docusate was examined using Vero cells with the neutral red uptake assay described by Schmidt and Korba (2000). Culture medium was removed from confluent Vero cells in 24-well plates. The cells were then washed once with PBS. One milliliter of culture medium containing Docusate or SDS at concentrations of 0, 0.001, 0.0025, 0.005, and 0.01% were added to each well. Cells were incubated at 37 °C for 1 h, 6 h, 2 and 3 days. At each time point, the medium was removed and cells washed with PBS. Five hundred microlitres of 0.01% neutral red (in PBS) was added to each well, and the samples were incubated at 37 °C for 30 min. The dye was then removed and the cells washed twice with 1 ml PBS per well. The dye was extracted by addition of 500 μl of 50% ethanol/1% glacial acetic acid in PBS to each well and incubated at room temperature for 15 min with gentle shaking at 120–150 rpm. Then 200 μl of extracted dye from each well was put into 96-well plate and the absorbance at 550 nm was read on an ELISA reader. [1]
Long-term cytotoxicity of docusate was also examined using trypan blue exclusion assay. Confluent Vero cells were washed with PBS and incubated with culture medium containing various concentrations of Docusate or SDS at 37 °C for 3 days. After culture medium was removed, the cells were washed once with PBS, trypsinized with Versene/1× trypsin and resuspended in 1 ml of 5% MEM culture medium. One hundred microlitres of cell suspension was mixed with 500 μl trypan blue solution. The viable cells were counted using a hemacytometer.

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Pre-treatment of cells [1]
Confluent Vero cells in 6-well plates were washed with PBS. One milliliter culture medium containing different concentrations of Docusate or SDS was added to each well and the cells were incubated at 37 °C for 1 h. Following pre-incubation of the cells with Docusate, two treatments were performed, one set of plates was directly infected with 100 pfu per well of HSV-2; another set of plates was washed three times with PBS, and then infected with 100 pfu per well of HSV-2. Both infections were incubated at 37 °C for 1 h with tilting every 10 min. After viral inoculum was removed, the infected cells were covered with methylcellullose for plaque assay.

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Effect of Docusate on HSV-infected cells [1]
Confluent Vero cells were washed with PBS and then infected with 100 pfu per well of HSV-2 at 37 °C for 1 h. Following removal of viral inoculum, infected cells were washed once with PBS and covered with 0.5% methylcellulose containing either Docusate or SDS at various concentrations for plaque assay.

Pregnant dams (n = 3–6 per group) from E11.5 through weaning were provided a with water bottles containing either vehicle control (0.5% carboxymethylcellulose; CMC) or CMC plus 31.25 μg/mL Docusate/DOSS, both prepared in autoclaved 18MOhm water to simulate DOSS use during human pregnancy. Stool softeners (e.g. Docusate, Colace, etc.), which are essentially made of Docusate/DOSS, are accepted as safe prescription drugs and used by the medical community as the standard of care to treat constipation during pregnancy and breastfeeding. CMC was used to increase palatability of the DOSS solution and has been used as a vehicle control in other obesogen dosing studies. CMC is also commonly found as a component in DOSS-containing stool softeners. The DOSS dosage (31.25 μg/mL) correlates to the dose received by a pregnant woman (88.5 kg average weight, with a daily Docusate sodium stool softener dosage at 5.6 mg/kg = 500 mg DOSS) assuming that an adult mouse weighing 25 g drinks about 4 mL per day. The DOSS dosage given to pregnant dams (31.25 µg/mL) was more than 70-fold less than the no-observed-adverse-effect-level (NOAEL) for maternal DOSS toxicity and teratogenicity set for mice and rats (400 mg/kg) previously. The timing of exposure was based on the incidence of constipation in women becoming elevated from mid-gestation onward. Stock bottles of each solution were prepared as needed (usually weekly). A 1% CMC solution in autoclaved water and a 62.5 μg/mL solution of DOSS in autoclaved water were prepared and then mixed together in a ratio of 1:1 to achieve the final desired concentration for treatment. Autoclaved water was used to dilute the 1% CMC solution 1:1 to achieve the desired concentration of 0.5% CMC for the vehicle control. Only litters with 5–8 pups were used for analysis to control for nutrient availability in utero and potential effects of DOSS on litter size. Previous studies indicated that there was no adverse effect on litter size using more than 70-fold higher doses of DOSS. Dosing experiments were repeated three times and control vs. treatment animals from different experiments were grouped for analyses. Investigators were blinded as to the control and treated group samples.

Effects During Pregnancy and Lactation
◈ What is docusate sodium?
Docusate sodium is a stool softener that has been used to treat constipation (when a person passes less than three bowel movements a week or has trouble with bowel movements). Docusate sodium is found in many products. Some brand names are Colace®, Correctol®, Docusate®, Surfak®, and Soflax®.Sometimes when people find out they are pregnant, they think about changing how they take their medication, or stopping their medication altogether. However, it is important to talk with your healthcare providers before making any changes to how you take this medication. Your healthcare providers can talk with you about the benefits of treating your condition and the risks of untreated illness during pregnancy.
◈ I take docusate sodium. Can it make it harder for me to get pregnant?
It is not known if docusate sodium can make it harder to get pregnant.
◈ Does taking docusate sodium increase the chance for miscarriage?
Miscarriage is common and can occur in any pregnancy for many different reasons. Studies have not been done to see if docusate sodium increases the chance for miscarriage. In general, very little docusate sodium is expected to get into the blood stream where it can then reach the developing pregnancy.
◈ Does taking docusate sodium increase the chance of birth defects?
Every pregnancy starts out with a 3-5% chance of having a birth defect. This is called the background risk.Available information suggests that, when used as directed, docusate sodium is unlikely to increase the chance of birth defects above the background risk.
◈ Does taking docusate sodium in pregnancy increase the chance of other pregnancy-related problems?
When used in recommended doses, docusate sodium is unlikely to cause problems during pregnancy, such as preterm delivery (birth before week 37) or low birth weight (weighing less than 5 pounds, 8 ounces [2500 grams] at birth).Using more than the recommended amount of docusate sodium can lower the levels of magnesium in a person’s blood. There is one reported case of low magnesium levels in a newborn that was linked to overuse of docusate sodium by the person who was pregnant. The baby’s main symptom was jitteriness, which went away by the second day of life. There have been no reported problems linked to the use of recommended amounts of docusate sodium in pregnancy.
◈ Does taking docusate sodium in pregnancy affect future behavior or learning for the child?
Studies have not been done to see if docusate sodium can cause behavior or learning issues for the child.
◈ Breastfeeding while taking docusate sodium:
The use of docusate sodium while breastfeeding has not been studied. Docusate sodium is not well-absorbed from the gastrointestinal tract and is unlikely to be found in breastmilk in large amounts. There was one report of diarrhea in an infant who was exposed to docusate sodium and another medication called danthron through breastmilk. There have been no other reports of problems in children exposed to docusate sodium during breastfeeding. If you suspect the baby has any symptoms (such as diarrhea), contact the child’s healthcare provider. Be sure to talk to your healthcare provider about all your breastfeeding questions.
◈ If a male takes docusate sodium, could it affect fertility (ability to get partner pregnant) or increase the chance of birth defects?
Studies have not been done to see if docusate sodium could affect male fertility or increase the chance of birth defects above the background risk. In general, exposures that fathers or sperm donors have are unlikely to increase risks to a pregnancy. For more information, please see the MotherToBaby fact sheet Paternal Exposures at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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23673837 rat LD50 oral 1900 mg/kg Journal of the Society of Cosmetic Chemists., 13(469), 1962
23673837 rat LD50 intraperitoneal 590 mg/kg VASCULAR: STRICTIRA; CJAMGES OM VESSE;S Bromatologia i Chemia Toksykologiczna., 7(161), 1974
23673837 mouse LD50 oral 2643 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY) Drug and Chemical Toxicology., 1(89), 1977/1978
23673837 mouse LD50 intravenous 60 mg/kg Journal of the American Pharmaceutical Association, Scientific Edition., 38(428), 1949

[1]. Preclinical evaluation of docusate as protective agent from herpes simplex viruses. Antiviral Res. 2001 Oct;52(1):25-32.

[2]. Increased adiposity, inflammation, metabolic disruption and dyslipidemia in adult male offspring of DOSS treated C57BL/6 dams. Sci Rep. 2019 Feb 6;9(1):1530.

Sodium docusate is an organic sodium salt.
Docusate Sodium is the sodium salt of docusate, a dioctyl salt and an emollient laxative with stool-softening activity. Docusate decreases surface tension and emulsification of fecal matter and allows water to penetrate and mix with stool. As a result, it softens the stool.
All-purpose surfactant, wetting agent, and solubilizer used in the drug, cosmetics, and food industries. It has also been used in laxatives and as cerumenolytics. It is usually administered as either the calcium, potassium, or sodium salt.
See also: Docusate (has active moiety); Docusate Sodium; Sennosides (component of); Benzocaine; docusate sodium (component of) ... View More ...

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Prevention of sexually transmitted infections (STIs) is key to public health efforts to control these diseases. An effective vaginal microbicide could provide topical, broad-spectrum prevention against the transmission of several STI pathogens. Docusate is a sulfated surfactant and, as such, may inactivate viral pathogens by disrupting viral envelopes and/or denaturing/disassociating proteins. Accordingly, the in vitro efficacy and toxicity of docusate (dioctyl sodium sulfosuccinate; Zorex; Meditech Pharmaceuticals, Inc., Scottsdale, Arizona) against herpes simplex viruses (HSV) were evaluated. Docusate was effective in vitro against wild type and drug-resistant strains of HSV type 1 and 2 with EC(90-100) (effective concentration giving 90-100% virus yield reduction) of approximately 0.005% (w/v). Sodium dodecyl sulfate (SDS) was equipotent, however, docusate was somewhat less toxic to uninfected Vero cells compared with SDS after 2 days incubation (docusate CC(50) approximately 0.01% vs. SDS approximately 0.005%). The cytotoxicity profiles of docusate were time- and dose-dependent and thus associated with the frequency of use. Kinetics of inactivation examined by pre-mixing virus and drug in a time-course experiment demonstrated that docusate could reach its EC(90-100) within 30 min. Docusate pretreatment of cells was associated with a 45% reduction in infectivity of those cells, despite a triple washing procedure. Once infected, an approximate 30% plaque reduction was observed with treatment. [1]

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Surfactants/detergents are an important class of vaginal microbicides for the control of STIs. These products destroy microorganisms by dissolving their outer membrane and limiting infectivities. The studies described here were designed to examine the virucidal activity and potential role of Docusate as a topical microbicide for the prevention of STIs or for the topical treatment herpes simplex virus-induced lesions. A recent report (van Damme L., Advances in topical microbicides, Presented at the XIII International AIDS Conference, 9–14 July, 2000, Durban, South Africa) stemming from a study of nonoxynol-9 as a potential vaginal microbicide has raised concerns about the clinical utility of surfactants because of an apparent increase in the rate of transmission of 50% in nonoxynol-9 users, with a direct correlation between frequency of use and risk of HIV transmission. Earlier analyses of this study did not suggest a significant clinical toxicity problem from this agent. However, the possibility that cytotoxicity potentiated HIV transmission has to be considered. SDS inactivates a broad-spectrum of viruses and STIs (Howett et al., 1999, Krebs et al., 1999, Krebs et al., 2000), and has been proposed as a possible topical microbicide. Accordingly, the efficacy of SDS was studied in parallel.
We found that docusate was highly effective against both HSV-1 and HSV-2 infection at a dose of 0.005% (w/v), including acyclovir and forscarnet resistant mutants with EC90–100 approximately 0.005%. Although this concentration was also effective for SDS, SDS was toxic to cells, causing 50% cell death. This result indicates that docusate is less toxic than SDS. As shown in the kinetics of inactivation for HSV-2 (Fig. 2), this concentration achieves EC90–100 between 30 min and 1 h. Docusate was earlier observed to be effective against a number of other microbes, such as respiratory syncytial virus, influenza virus, bacteria, and fungi (unpublished data from Meditech Pharmaceuticals, Inc.). We speculate that it should be active against all enveloped viruses, and possibly nonenveloped viruses as well because surfactants can denature/disassociate proteins, as has been demonstrated for SDS, which can inactivate nonenveloped human papillomavirus (Howett et al., 1999).
We demonstrated that Docusate was effective inactivating HSV-2 and blocking virus adsorption/or replication in pre-treated cells even after docusate was removed by three washes. Although the nature of the interaction between cells and drug is not known, we speculate that a portion of docusate was bound/adsorbed to the cell surface or taken up by the cells. Slight antiviral effect of docusate was observed on cells that were already infected with HSV, suggesting the possibility of limited cell uptake of docusate.
Cytotoxicity is a common problem for surfactants used as topical microbicides owing to indiscrimination of cellular membranes and viral envelopes. This is associated with frequency of use. We have shown with both Docusate and SDS that this phenomenon is time- and dose-dependent. We demonstrated that the viability of cells cultured in vitro was dependent on microbicide concentrations and exposure duration. During short-term exposure (1 h) both docusate and SDS were minimally toxic at concentrations tested. During long-term exposure, docusate caused decreased cell viability with increased concentrations. A concentration of 0.01% was found to be 50–100% toxic to Vero cells after prolonged incubation for 3 days. The cytotoxicity of docusate was less toxic than SDS. CC50 of docusate for 2 days incubation was approximately 0.01% and that of SDS was ∼0.005%. Krebs et al. (1999) showed that SDS was the least toxic among N-9, C31G, and SDS. This may suggest that docusate is less toxic than N-9 and C31G.
Our studies demonstrate that docusate has favorable in vitro characteristics warranting consideration for use as a potent topical microbicide in the prevention of STIs. The clinical impact of toxicity potential of surfactants in this setting remains to be determined. [1]

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In summary, this work provides evidence that DOSS acts as a developmental obesogen in vivo. This study highlights the need to develop effective DOSS detection methods for human biological samples and to further investigate the health effects associated with DOSS exposure in human populations. Upon DOSS treatment, results from male offspring varied in magnitude in that some animals exhibited a more exaggerated fat accumulation effect and others demonstrated higher/lower adiposity levels, including circulating leptin. Natural individual differences (e.g. social ranking) may be a large contributing factor to the variance observed. Other factors that may have contributed to the variance include: differences in maternal water consumption resulting in altered effective DOSS dosage levels, differences in food consumption and physical activity of pups, or seasonality of offspring birth. Additional studies are needed to determine the effect each factor contributed to the variance. Nevertheless, given the observed metabolic disruptions in male offspring from dams treated with DOSS, the use, safety and prescription of Docusate stool softener and possible significant dietary sources of DOSS during human pregnancy should face further scrutiny.[2]

C20H37NAO7S

444.5584

444.215

C, 54.04; H, 8.39; Na, 5.17; O, 25.19; S, 7.21

577-11-7

10041-19-7 (acid);128-49-4 (calcium);577-11-7 (sodium);7491-09-0 (potassium); 15968-85-1 (aluminum);

23673837

White to off-white solid powder

1.1

173-179 °C(lit.)

199ºC

4.89

0

7

18

29

546

0

CCCCC(CC)COC(=O)CC(C(=O)OCC(CC)CCCC)S(=O)(=O)[O-].[Na+]

APSBXTVYXVQYAB-UHFFFAOYSA-M

InChI=1S/C20H38O7S.Na/c1-5-9-11-16(7-3)14-26-19(21)13-18(28(23,24)25)20(22)27-15-17(8-4)12-10-6-2;/h16-18H,5-15H2,1-4H3,(H,23,24,25);/q;+1/p-1

sodium;1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate

Docusate sodium; Dioctylal; Diotilan; Disonate; Molatoc; Regutol; Velmol; ...; 577-11-7; Dioctyl sodium sulfosuccinate (DOSS)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 20 mg/mL (~44.99 mM)

Solubility in Formulation 1: 50 mg/mL (112.47 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)